Characterization of a Novel Bacteriophage Endolysin (LysAB1245) With Extended Lytic Activity Against Distinct Capsular Types Associated With Acinetobacter Baumannii Resistance

Abstract

Capsular polysaccharides are considered as major virulence factors associated with the ability of multidrug-resistant (MDR) Acinetobacter baumannii to cause severe infections. In this study, LysAB1245, a novel bacteriophage-encoded endolysin consisting of a lysozyme-like domain from phage T1245 was successfully expressed, purified, and evaluated for its antibacterial activity against distinct capsular types associated with A. baumannii resistance. The results revealed a broad spectrum activity of LysAB1245 against all clinical MDR A. baumannii isolates belonging to capsular type (KL) 2, 3, 6, 10, 47, 49, and 52 and A. baumannii ATCC 19606. At 2 h following the treatment with 1.7 unit/reaction of LysAB1245, more than 3 log reduction in the numbers of bacterial survival was observed. In addition, LysAB1245 displayed rapid bactericidal activity within 30 min (nearly 3 log CFU/mL of bacterial reduction). Thermostability assay indicated that LysAB1245 was stable over a broad range of temperature from 4 to 70°C, while pH sensitivity assay demonstrated a wide range of pH from 4.5 to 10.5. 

Introduction

Recently, an outbreak of A. baumannii infections in coronavirus disease 2019 (COVID-19) patients has been reported [1–3], resulting in increased morbidity and mortality rates as well as high treatment costs. This emerging pathogen is responsible for several healthcare-associated infections, such as bacteremia, ventilator-associated pneumonia, urinary tract infections, burn and wound infections, and meningitis [4–7]. Nosocomial A. baumannii infections usually correlate with the production of capsular polysaccharide (CPS), which plays an important role in bacterial pathogenesis by protecting from environmental stresses, antimicrobial penetration, and host immune responses [8,9]. In 2019, more than 100 distinct capsular types (KL) of A. baumannii were discovered, with the variations in K unit structures and sugar composition [10]. In a previous study, three capsular genotypes, including KL6, 10, and 47, showed a frequency more than 10% among A. baumannii isolates from three tertiary care hospitals in Thailand [11]. 

Materials and methods

Bacteria, bacteriophage, and culture conditions

The bacterial strains, phage, plasmids, and primers used in this study are listed in Table 1. All MDR A. baumannii strains belonged to sequence type 2 with different major capsular types, including KL2, 3, 6, 10, 47, 49, and 52. All bacteria were inoculated in Luria Bertani (LB) broth or LB agar (Difco Laboratories, Detroit, MI, USA) at 37°C and maintained in 20% glycerol (v/v) at -80°C for long-term storage. This study was approved by the Human Research Ethics Committee (HREC) of the Faculty of Medicine at Prince of Songkla University (reference number: 64–284–14–1).

Results 

Characterization of endolysin LysAB1245

The endolysin gene of phage T1245, named LysAB1245 contains 558 base pairs and 185 amino acids. BLAST analysis showed that the LysAB1245 gene had 98.92% sequence similarity to putative chitinase-like endolysin from Acinetobacter phage phiAB6 (accession no. YP_009288673.1). The results of conserved domain analysis using the Pfam database revealed that amino acids of LysAB1245 contain lysozyme-like (N-acetyl-β-D-muramidase) domains between residues 79 and 136, which are the catalytic domains of LysAB1245. The catalytic activities of purified endolysin LysAB1245 are attributable to glycosidases that cleave β-1,4-N-acetyl-D-glucosamine bonds between N-acetylmuramic acid and N-acetylglucosamine in glycan chains [32]. 

Discussion

Antimicrobial activity of LysAB1245

Furthermore, we examined the antimicrobial activity of LysAB1245 against 20 MDR A. baumannii isolates and ATCC 19606. The MIC value of LysAB1245 was 4.21 μg/mL (0.1 unit/reaction) for all MDR A. baumannii isolates and A. baumannii ATCC 19606. The lowest concentration of LysAB1245 with bactericidal activity was 4.21 μg/mL, which was identical to the MIC value (Table 2). In general, the peptidoglycan structure of Gram-negative bacteria is highly conserved. Therefore, the conservation of the peptidoglycan structure among the tested isolates, which serves as the target site of endolysin, might have resulted in the same MIC values of LysAB1245.

Conclusions

In the present study, the endolysin LysAB1245 from Acinetobacter phage T1245 was successfully expressed and purified using an automated protein synthesis system with high-purity target proteins. A novel purified LysAB1245 exhibited a broad lytic spectrum activity against MDR A. baumannii isolates, which belong to various major capsular types. Additionally, LysAB1245 displayed rapid bactericidal activity and stability under various pH and temperature conditions. This work elucidated a potential of LysAB1245 as a new potential therapeutic agent towards the management of MDR A. baumannii infections.

Citation: Soontarach R, Srimanote P, Arechanajan B, Nakkaew A, Voravuthikunchai SP, Chusri S (2024) Characterization of a novel bacteriophage endolysin (LysAB1245) with extended lytic activity against distinct capsular types associated with Acinetobacter baumannii resistance. PLoS ONE 19(1): e0296453. https://doi.org/10.1371/journal.pone.0296453

Editor: Iddya Karunasagar, Nitte University, INDIA

Received: August 8, 2023; Accepted: December 11, 2023; Published: January 2, 2024

Copyright: © 2024 Soontarach et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This research was funded by the National Research Council of Thailand (Grant No. N41A640071) and the Postdoctoral Fellowship from Prince of Songkla University, Thailand. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0296453#abstract0